In certain bacteria, a unique tRNA modification occurs in which the sulfur of a 5-methylaminomethyl-2-thiouridine nucleoside in Glu,Lys,Gln tRNA is replaced with selenium (Se). This specific Se incorporation process is catalyzed by the enzyme, tRNA 2-selenouridine synthase (tRNA 2-SeUS). One of the known biosynthetic steps involves the synthesis of selenophosphate (the universal biological Se donor for the tRNA modification and for incorporation of Se into proteins) from ATP and selenide by selenophosphate synthetase. The specific mechanism for the tRNA modification is not known. In a four-step procedure, we have extended previous work and partially purified 2-selenouridine synthase activity. Enzyme activity appears to require more than one protein, and the greatest increase in specific tRNA 2-SeUS activity is associated with enrichment of low molecular mass proteins (less than 30 kDa). Interestingly, a few ribosomal proteins, L1, L3, and S2, also co-purify with the selenouridine synthase activity. Selenium-containing tRNAs that have nucleosides distinct from 5-methylaminomethyl-2-selenouridine were detected in the hyperthermophilic organism, Methanococcus jannaschii, and in a number of cell lines, including HeLa and Jurket T cells (collaboration with Dr. Kuan-Teh Jeang). Elucidation of their structures using liquid chromatography mass spectrometry techniques will be carried out in collaboration with Professor James A. McCloskey at the University of Utah, who is a leader in the field of mass spectrometry of modified nucleosides in nucleic acids.